Name: genetic manipulation of cerebellar granule neurons in vitro and in vivo to study neuronal morphology and migration
Uploaded: 2014-03-17T17:45:00
Description: Watch this Scientific Journal Video about Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration at JoVE.com

我们感谢N. Schwedhelm - 杜美尔优秀的技术援助，三锤和S. Papiol帮助与统计分析。我们的工作是由马普学会，德意志研究联合会，该中心的纳米显微镜，及脑分子生理学（CNMPB），哥廷根，德国和由GGNB初级组助学金哥廷根大学的资助。

小脑颗粒神经元可以从产后当天（P）5小鼠幼崽或P6幼鼠准备。我们遵循的协议，由Bilimoria医师和他的同事描述的，它采用了有丝分裂抑制剂，以选择用于有丝分裂后小脑颗粒神经元13。 伦理语句： 根据批准的“Verbraucherschutz UND Lebensmittelsicherheit”下萨克森州，德国的动物协议所有涉及活的动物实验已经进行的。 体外测定： <p class="jov...

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优点和在体外和体内方法所描述的限制： 从小鼠和大鼠培养的小脑颗粒神经元也同样适用以及形态学分析。由于一只老鼠小脑的大尺寸，CNGS从幼鼠的产量超过了小鼠幼崽3至4倍。除小脑颗粒神经元，皮质和海马神经元可被用作培养系统为好。磷酸钙方法导致低（0.01-5％）的转染效率，这是需要的，分析单个神经元的形态。替代亲脂转染方法可以用于为好，但过?...

小脑是一个很好的系统来研究轴突生长和迁移的机制。自1神经科学的曙光小脑已经解剖研究的主题。现代显微镜和免疫组化技术已显著扩大，由圣地亚哥，拉蒙和卡哈尔2-4细化初始的发现。小鼠遗传学和分子生物学研究发现重要的增长和转录因子在小脑发育的控制，从而导致需要对不同类型的神经元，包括小脑颗粒神经元（小脑颗粒神经元）5-7的正确接线关键事件的更深入的了解。 小脑是显影后脑8的菱1的衍生物。菱形唇，它是4 个脑室的屋顶的一部分，引起的小脑颗粒神经元祖细胞，这将构成最大量的神经元群中的成人小脑9。下面的喙迁移，他们定居在小脑原基。在这里，颗粒神经元前体有丝分裂导致外颗粒层（EGL），其中发生在出生后啮齿动物的急剧扩张。来自东瀛，神经元开始通过分子层（ML）向内迁移，过去的浦肯野细胞层，最终定居在内部颗粒层（IGL 2）。在这个迁徙的过程中，他们获得一个双极形状有两个轴突延伸到ML。在进一步迁移，细胞体迁移远离轴突和两个进程融合以形成一个分叉的，T形的轴突10。随后，这些轴突束状和被称为平行纤维。已入驻IGL，小脑颗粒神经元树突生长，形成树枝状的爪子，建立突触苔藓纤维。审查基本过程中的显影小脑，组合在体外和体内 approach允许进行可靠的结果和结论。 小脑颗粒神经元不仅是小脑而是整个大脑的最大量的神经元，并且可以被培养，以高纯度11-13。在文化方面，这个高度同质化的神经元群变得迅速和有丝分裂后获得一个极形态，易于识别轴突和树突。培养的小脑颗粒神经元已被证明是研究神经发育的各个方面，包括祖细胞增殖，分化，轴突和树突发育，神经细胞迁移，凋亡及电生理特性（14-19和许多其他人）非常有用。利用基因操作的扩大培养的小脑颗粒神经元的通用性，并允许进行进一步机械洞察上述事件。使用低效率的磷酸钙或亲脂性的方法，其次是免疫细胞化学与极性标记或软件支持的分析facili培养神经元的转染塔特斯单个神经元，例如在一个密集的神经元文化形态的评估。用这种方法，在轴突或枝晶生长兴趣蛋白质的作用，可以研究20-25,26-28。然而，这种文化体系是分析神经细胞迁移的迁移是非常有限的高密度培养的那么有用，并需要共同培养。 在体外分析轴突和树突生长还允许一个信号传导途径的利用RNA干扰（I）的组合相互连接的蛋白质的检测，过度表达或小分子。 建立在轴突和树突生长调控或神经细胞迁移的目的蛋白的相关性， 在体内电穿孔（IVE）技术允许用于分析在显影小脑皮层。由于其在啮齿类动物小脑发育延伸的方式进入第一产后2周的事实，小脑表示accessib乐大脑结构的遗传操作研究发展轴突和树突，神经元迁移，突触和凋亡20-24,29,30,26,27,31-34。此外，该模型体系也为神经元发育的需要完整的小脑皮层如轴突路径，布线和神经元和神经胶质细胞的相互作用两者合计的连接等方面有用的，该协议提供了在体外和体内技术来解决有关神经元的形态发生和迁移互补的方法。

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			<td height="21" style="height:21px;width:160px;">DMEM</td>
			<td style="width:125px;">Gibco</td>
			<td style="width:88px;">11960-044</td>
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			<td height="21" style="height:21px;width:160px;">BME</td>
			<td style="width:125px;">Gibco</td>
			<td style="width:88px;">41010-026</td>
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			<td height="21" style="height:21px;width:160px;">Insulin</td>
			<td style="width:125px;">Sigma-Aldrich</td>
			<td style="width:88px;">Si-1-4011</td>
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			<td height="21" style="height:21px;width:160px;">Poly-L-Ornithine</td>
			<td style="width:125px;">Sigma-Aldrich</td>
			<td style="width:88px;">P-2533</td>
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			<td height="25" style="height:25px;width:160px;">CaCl2</td>
			<td style="width:125px;">Appli-Chem</td>
			<td style="width:88px;">A3652</td>
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			<td height="42" style="height:42px;width:160px;">Isofluorane</td>
			<td style="width:125px;">Actavis Deutschland</td>
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			<td height="22" style="height:22px;width:160px;">Tissue-Tek OCT</td>
			<td style="width:125px;">Sakura</td>
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			<td height="44" style="height:44px;width:160px;">Material Name</td>
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			<td style="width:88px;">Catalogue Number</td>
			<td style="width:168px;">Comments (optional)</td>
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			<td height="43" style="height:43px;width:160px;">ECM 830 and tweezertrodes</td>
			<td style="width:125px;">Harvard Apparatus</td>
			<td style="width:88px;"></td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:160px;">Epifluorescence microscope and camera</td>
			<td style="width:125px;">Nikon</td>
			<td style="width:88px;"></td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:160px;">SP2 confocal microscope</td>
			<td style="width:125px;">Leica</td>
			<td style="width:88px;"></td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:160px;">ImageJ</td>
			<td style="width:125px;">NIH</td>
			<td style="width:88px;"></td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:160px;">Imaris 7.4.2</td>
			<td style="width:125px;">Bitplane, Inc.</td>
			<td style="width:88px;"></td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:160px;">GraphPad Prism</td>
			<td style="width:125px;">GraphPad Software, Inc.</td>
			<td style="width:88px;"></td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:160px;">MS Excel</td>
			<td style="width:125px;">Microsoft</td>
			<td style="width:88px;"></td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:160px;">Loading tip 1-200 &#181;l</td>
			<td style="width:125px;">Costar</td>
			<td style="width:88px;">4853</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:160px;">Pipette tip 200 &#181;l</td>
			<td style="width:125px;">Sarstedt</td>
			<td style="width:88px;">70.760.502</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:160px;">Microlance 3 needle, 30 gauge</td>
			<td style="width:125px;">BD</td>
			<td style="width:88px;">302200</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:160px;">50 &#181;l gastight Syringe 1705</td>
			<td style="width:125px;">Hamilton</td>
			<td style="width:88px;"></td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:160px;">Glass coverslips&#160;</td>
			<td style="width:125px;">Thermo Scientific Menzel Glaeser</td>
			<td>CB00120RA1</td>
			<td></td>
		</tr>
	</tbody>
</table>

genetic manipulation of cerebellar granule neurons in vitro and in vivo to study neuronal morphology and migration

在大脑发育事件包括神经元的形态发生和迁移是高度协调的过程， 在体外和体内分析，以便作深入刻画，以确定参与这些活动的途径。小脑颗粒神经元（小脑颗粒神经元），这些来自发展中国家小脑派生是一个理想的模型系统，允许进行形态学分析。在这里，我们描述了如何将遗传操纵小脑颗粒神经元的方法，以及如何学习axono和树突单个神经元。用这种方法RNA干扰，过表达或小分子的效应可以比较来控制神经元。另外，啮齿动物小脑皮层是体内系统，由于其优越的出生后发育的方便。我们还提出了一个在体内电穿孔技术，基因操作的开发小脑，并说明后续小脑分析评估神经元形态的ND迁移。

分析小脑颗粒神经元的响应不同培养条件的形态，我们转染对DIV 0的神经元，如上所述。转染后，我们把一组神经元的充分培养基（BME，10％小牛血清，2mM PSG，25mM的KCl）中，另一组为最小的含有培养基的胰岛素（BME，25mM的葡萄糖，2mM的PSG，10微克/毫升胰岛素）。我们使用GFP抗体在DIV 1,2进行的神经元免疫细胞化学，和3，随后通过测量轴突和树突为组1和轴突只为一组2。由于血清和KCl，提供生长因?...

Watch this Scientific Journal Video about Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration at JoVE.com

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

神经元的形态发生和迁移的基本正常的大脑发育至关重要的事件。在这里，我们描述的方法来操纵基因培养的小脑颗粒神经元和小脑发展的形态和神经元的迁徙特性的评估。

小脑颗粒神经元的遗传操作<em&gt;体外</em&gt;和<em&gt;在体内</em&gt;为研究神经元形态和迁移

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

Research

JoVE Journal

Neuroscience

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Automated Sholl Analysis of Digitized Neuronal Morphology at Multiple Scales

肖尔自动数字化的神经元形态分析，多尺度

Neuronal morphology plays a significant role in determining how neurons function and communicate1-3.  Specifically, it affects the ability of neurons to ...

科研

神经科学

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

嗅觉感官神经元的慢病毒介导的遗传操作和可视化在体内</em

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the ...

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

使用调频染料培养的小脑颗粒神经突触小泡池补充的定量分析

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

谷氨酸和GABA快速微离子导入：一个有用​​的工具，调查突触整合

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Nucleofection of Rodent Neuroblasts to Study Neuroblast Migration In vitro

Nucleofection of Rodent Neuroblasts to Study Neuroblast Migration In vitro

鼠神经母细胞核转染到研究神经细胞迁移<em&gt;在体外</em

The subventricular zone (SVZ) located in the lateral wall of the lateral ventricles plays a fundamental role in adult neurogenesis. In this restricted ...

Laser Nanosurgery of Cerebellar Axons In Vivo

Laser Nanosurgery of Cerebellar Axons In Vivo

小脑轴突的激光Nanosurgery<em&gt;在体内</em

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

诱导星形胶质细胞受体的可塑性由神经元的放电频率操作

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

嗅觉系统作为模型来研究轴突生长的模式和形态<em&gt;在体内</em

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

产后的小脑颗粒细胞迁移利用共聚焦肉眼离体成像

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then ...

Analyzing Dendritic Morphology in Columns and Layers

分析树突状形态的列和图层

In many regions of the central nervous systems, such as the fly optic lobes and the vertebrate cortex, synaptic circuits are organized in layers and ...